Decellularized human periodontal ligament for periodontium regeneration

Abstract

Regenerating the periodontal ligament (PDL) is a crucial factor for periodontal tissue regeneration in the presence of traumatized and periodontally damaged teeth. Various methods have been applied for periodontal regeneration, including tissue substitutes, bioactive materials, and synthetic scaffolds. However, all of these treatments have had limited success in structural and functional periodontal tissue regeneration. To achieve the goal of complete periodontal regeneration, many studies have evaluated the effectiveness of decellularized scaffolds fabricated via tissue engineering. The aim of this study was to fabricate a decellularized periodontal scaffold of human tooth slices and determine its regeneration potential. We evaluated two different protocols applied to tooth slices obtained from human healthy third molars. The extracellular matrix scaffold decellularized using sodium dodecyl sulfate and Triton X-100, which are effective in removing nuclear components, was demonstrated to preserve an intact structure and composition. Furthermore, the decellularized scaffold could support repopulation of PDL stem cells near the cementum and expressed cementum and periodontal-ligament-related genes. These results show that decellularized PDL scaffolds of human teeth are capable of inducing the proliferation and differentiation of mesenchymal stem cells, thus having regeneration potential for use in future periodontal regenerative tissue engineering.

Fig 1. Comparison of different decellularization protocols for periodontal tissue in human tooth slices.

(A) Residual nuclei (arrowheads) are indicated by hematoxylin and eosin (H&E) staining (a–c) and fluorescence staining with 4',6-diamidino-2-phenylindole (DAPI) (d–f). More cell nuclei were removed in Protocol II (PII) than Protocol I (PI) after decellularization in H&E and DAPI staining. (B) Residual total DNA contents after decellularization of the periodontal ligament (PDL). The normality of the data was evaluated using the Shapiro-Wilk test (p<0.05). *p<0.05 in Kruskal-Wallis test followed by the post-hoc Bonferroni test (Bonferroni correction; p<0.017). (C) Scanning electron microscopy images of the different protocol groups. Arrowheads indicate remaining Sharpey’s fibers in the PDL. D, dentin. Scale bars: 20 μm in A(a–c), 50 μm in A(d–f), and 20 μm in C.

Fig 2. Immunohistochemistry (IHC) staining of collagen I (Col I), collagen XII (Col XII), and fibronectin in the decellularized PDL of tooth slices.

Scale bars: 20 μm in a–i.

Fig 3. Characterization of decellularized human PDL periodontal ligament (dHPDL) recellularized with PDL stem cells (PDLSCs).

(A) Cell viability for PII after recellularization. PDLSCs repopulated onto dHPDL when applying PII were viable compared with the control group. The Mann-Whitney U test (*p<0.05) was used to assess cell viability. (B) PDLSCs seeded onto decellularized tooth slices in PII repopulated and infiltrated toward the cementum (black arrows) in H&E and Masson’s trichrome (MT) staining. PDLSCs expressed osteocalcin (OC) and cementum protein 23 (CP23) near the cementum (red arrows) in recellularized human tooth slices in IHC staining. C, cementum. Scale bars: 20 μm.

Fig 4. Gene expression in the recellularized human PDL group analyzed using the quantitative real-time reverse-transcription polymerase chain reaction.

PDLSCs were seeded in the control group or in the decellularized group using PII. *p<0.05 for alkaline phosphatase (ALP) in Mann-Whitney U test; *p<0.05 for CP23 and OC in Student’s t-test.